Bottom Line:
The free energy difference between β- and α-anomers (ΔGβ-α) of all d-stereoisomers in water were compared to experimental values with a good agreement.The relative binding free energies (ΔΔGbind) were calculated and, where available, compared to experimental values, approximated from Km values.The results suggest that a similar approach could be applied to study promiscuity of other enzymes.

Affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.

ABSTRACTThe flavoenzyme pyranose dehydrogenase (PDH) from the litter decomposing fungus Agaricus meleagris oxidizes many different carbohydrates occurring during lignin degradation. This promiscuous substrate specificity makes PDH a promising catalyst for bioelectrochemical applications. A generalized approach to simulate all 32 possible aldohexopyranoses in the course of one or a few molecular dynamics (MD) simulations is reported. Free energy calculations according to the one-step perturbation (OSP) method revealed the solvation free energies (ΔGsolv) of all 32 aldohexopyranoses in water, which have not yet been reported in the literature. The free energy difference between β- and α-anomers (ΔGβ-α) of all d-stereoisomers in water were compared to experimental values with a good agreement. Moreover, the free-energy differences (ΔG) of the 32 stereoisomers bound to PDH in two different poses were calculated from MD simulations. The relative binding free energies (ΔΔGbind) were calculated and, where available, compared to experimental values, approximated from Km values. The agreement was very good for one of the poses, in which the sugars are positioned in the active site for oxidation at C1 or C2. Distance analysis between hydrogens of the monosaccharide and the reactive N5-atom of the flavin adenine dinucleotide (FAD) revealed that oxidation is possible at HC1 or HC2 for pose A, and at HC3 or HC4 for pose B. Experimentally detected oxidation products could be rationalized for the majority of monosaccharides by combining ΔΔGbind and a reweighted distance analysis. Furthermore, several oxidation products were predicted for sugars that have not yet been tested experimentally, directing further analyses. This study rationalizes the relationship between binding free energies and substrate promiscuity in PDH, providing novel insights for its applicability in bioelectrochemistry. The results suggest that a similar approach could be applied to study promiscuity of other enzymes.

pcbi-1003995-g005: Distributions of all five improper dihedrals (ID) for the MD simulations in water and in protein.For system (top five panels), the distributions of the ID are derived from the single 100 ns MD simulation, which sampled all 32 possible stereoisomers (also compare to Fig. 4, left two panels). For system PDH-SUG (pose A) (middle five panels), and for system PDH-SUG (pose B) (lowest five panels), the occurrences as observed in the four selected MD simulations were arithmetically averaged. Coloring scheme according to Fig. 2A.

Mentions:
(A) System SUGa: improper dihedrals (ID) at stereocenters C1–C5 were turned off. Numbers indicate the name of the C-atom and the ID-position within the 5-digit ID code in Tables 1 and 3. Colors are in agreement with the coloring schemes in Figs. 5–7: green (C1, ID1), yellow (C2, ID2), red (C3, ID3), blue (C4, ID4), and black (C5, ID5). (B) System SUGab: in addition to system SUGa, proper dihedral force constants (kφ) for the ring torsional dihedral angles were lowered according to the following coloring scheme: blue (two force constants from 2.09 to 0.418 kJ mol−1 and one from 5.92 to 1.05 kJ mol−1), red (one force constant from 5.92 to 1.05 kJ mol−1), and yellow (two force constants from 3.77 to 1.05 kJ mol−1). (C) System SUGabc: in addition to system SUGab, bond angle bending force constants (kθ) for the bond angles surrounding the ring atoms (C1–C5 and O) were lowered according to following coloring scheme: blue (one bond angle from 380 to 285 kJ mol−1), red (two bond angles from 320 to 285 kJ mol−1), yellow (three bond angles from 320 to 285 kJ mol−1).

pcbi-1003995-g005: Distributions of all five improper dihedrals (ID) for the MD simulations in water and in protein.For system (top five panels), the distributions of the ID are derived from the single 100 ns MD simulation, which sampled all 32 possible stereoisomers (also compare to Fig. 4, left two panels). For system PDH-SUG (pose A) (middle five panels), and for system PDH-SUG (pose B) (lowest five panels), the occurrences as observed in the four selected MD simulations were arithmetically averaged. Coloring scheme according to Fig. 2A.

Mentions:
(A) System SUGa: improper dihedrals (ID) at stereocenters C1–C5 were turned off. Numbers indicate the name of the C-atom and the ID-position within the 5-digit ID code in Tables 1 and 3. Colors are in agreement with the coloring schemes in Figs. 5–7: green (C1, ID1), yellow (C2, ID2), red (C3, ID3), blue (C4, ID4), and black (C5, ID5). (B) System SUGab: in addition to system SUGa, proper dihedral force constants (kφ) for the ring torsional dihedral angles were lowered according to the following coloring scheme: blue (two force constants from 2.09 to 0.418 kJ mol−1 and one from 5.92 to 1.05 kJ mol−1), red (one force constant from 5.92 to 1.05 kJ mol−1), and yellow (two force constants from 3.77 to 1.05 kJ mol−1). (C) System SUGabc: in addition to system SUGab, bond angle bending force constants (kθ) for the bond angles surrounding the ring atoms (C1–C5 and O) were lowered according to following coloring scheme: blue (one bond angle from 380 to 285 kJ mol−1), red (two bond angles from 320 to 285 kJ mol−1), yellow (three bond angles from 320 to 285 kJ mol−1).

Bottom Line:
The free energy difference between β- and α-anomers (ΔGβ-α) of all d-stereoisomers in water were compared to experimental values with a good agreement.The relative binding free energies (ΔΔGbind) were calculated and, where available, compared to experimental values, approximated from Km values.The results suggest that a similar approach could be applied to study promiscuity of other enzymes.

Affiliation:
Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.

ABSTRACTThe flavoenzyme pyranose dehydrogenase (PDH) from the litter decomposing fungus Agaricus meleagris oxidizes many different carbohydrates occurring during lignin degradation. This promiscuous substrate specificity makes PDH a promising catalyst for bioelectrochemical applications. A generalized approach to simulate all 32 possible aldohexopyranoses in the course of one or a few molecular dynamics (MD) simulations is reported. Free energy calculations according to the one-step perturbation (OSP) method revealed the solvation free energies (ΔGsolv) of all 32 aldohexopyranoses in water, which have not yet been reported in the literature. The free energy difference between β- and α-anomers (ΔGβ-α) of all d-stereoisomers in water were compared to experimental values with a good agreement. Moreover, the free-energy differences (ΔG) of the 32 stereoisomers bound to PDH in two different poses were calculated from MD simulations. The relative binding free energies (ΔΔGbind) were calculated and, where available, compared to experimental values, approximated from Km values. The agreement was very good for one of the poses, in which the sugars are positioned in the active site for oxidation at C1 or C2. Distance analysis between hydrogens of the monosaccharide and the reactive N5-atom of the flavin adenine dinucleotide (FAD) revealed that oxidation is possible at HC1 or HC2 for pose A, and at HC3 or HC4 for pose B. Experimentally detected oxidation products could be rationalized for the majority of monosaccharides by combining ΔΔGbind and a reweighted distance analysis. Furthermore, several oxidation products were predicted for sugars that have not yet been tested experimentally, directing further analyses. This study rationalizes the relationship between binding free energies and substrate promiscuity in PDH, providing novel insights for its applicability in bioelectrochemistry. The results suggest that a similar approach could be applied to study promiscuity of other enzymes.